The human tumor virus, Epstein Barr Virus (EBV), encodes multiple microRNAs (miRNAs) that are expressed during latent infection in tumors. Two groups of miRNAs have been identified. One group is only expressed in transformed lymphocytes in Type III latency. The second group containing two clusters is produced from a complex family of transcripts, termed BARTs, from the BamHI A region of the genome. Importantly, these transcripts are highly produced in EBV-associated tumors but are expressed at low levels in infected B-lymphoid cell lines. Several of the EBV miRNAS are highly conserved with those identified in rhesus EBV with considerably greater homology than EBV/KSHV coding sequences. This conservation of sequences suggests that the miRNAs might target cellular messages. Based on these findings, this application will test the hypothesis that the EBV BART miRNAs target cellular genes to affect growth regulation. The proposed experiments will use a combination of bioinformatic, microarray, and proteomics to identify the cellular targets of the BART miRNAs. Cell lines that stably express the BART miRNAs will be produced to confirm expression and to determine the effects of the miRNAs on cellular growth properties. Multiple cellular signaling pathways are affected by EBV transforming proteins so potential modulation of these pathways by the BART miRNAs will also be examined. The effects of loss of miRNA expression in EBV infected cells will be assessed using antagomirs, sponges, and inhibitors of miRNA synthesis. Cellular miRNAs have been shown to be major factors in the development of cancer. This proposal will determine how the EBV miRNAs contribute to EBV-mediated oncogenesis. PUBLIC HEALTH RELEVANCE: Infection with Epstein-Barr virus contributes to the development of several important human malignancies through the effects of viral proteins on cell growth regulation. A new form of growth regulation has been discovered that involves small RNAs (miRNAs) that affect the function of cellular genes and contribute to cancer. EBV encodes multiple miRNAs that we have shown to be expressed at high levels in some EBV-associated cancers particularly nasopharyngeal carcinoma. The identification of cellular proteins that are targeted by the EBV miRNAs will increase our understanding of how EBV contributes to the development of cancer.